APH(3’)-Ie, an aminoglycoside-modifying enzyme discovered in a rabbit-derived Citrobacter gillenii isolate

Background Aminoglycoside-modifying enzymes (AMEs) play an essential role in bacterial resistance to aminoglycoside antimicrobials. With the development of sequencing techniques, more bacterial genomes have been sequenced, which has aided in the discovery of an increasing number of novel resistance mechanisms. Methods The bacterial species was identified by 16S rRNA gene homology and average nucleotide identity (ANI) analyses. The minimum inhibitory concentration (MIC) of each antimicrobial was determined by the agar dilution method. The protein was expressed with the pCold I vector in E. coli BL21, and enzyme kinetic parameters were examined. The whole-genome sequence of the bacterium was obtained via the Illumina and PacBio sequencing platforms. Reconstruction of the phylogenetic tree, identification of conserved functional residues, and gene context analysis were performed using the corresponding bioinformatic techniques. Results A novel aminoglycoside resistance gene, designated aph(3’)-Ie, which confers resistance to ribostamycin, kanamycin, sisomicin and paromomycin, was identified in the chromosome of the animal bacterium Citrobacter gillenii DW61, which exhibited a multidrug resistance phenotype. APH(3’)-Ie showed the highest amino acid identity of 74.90% with the functionally characterized enzyme APH(3’)-Ia. Enzyme kinetics analysis demonstrated that it had phosphorylation activity toward four aminoglycoside substrates, exhibiting the highest affinity (K m, 4.22 ± 0.88 µM) and the highest catalytic efficiency [k cat/K m, (32.27 ± 8.14) × 104] for ribomycin. Similar to the other APH(3’) proteins, APH(3’)-Ie contained all the conserved functional sites of the APH family. The aph(3’)-Ie homologous genes were present in C. gillenii isolates from different sources, including some of clinical significance. Conclusion In this work, a novel chromosomal aminoglycoside resistance gene, designated aph(3’)-Ie, conferring resistance to aminoglycoside antimicrobials, was identified in a rabbit isolate C. gillenii DW61. The elucidation of the novel resistance mechanism will aid in the effective treatment of infections caused by pathogens carrying such resistance genes.


Introduction
Since the discovery and isolation of the first aminoglycoside antibiotic, streptomycin, from soil bacteria in 1944 (Jones et al., 1944), aminoglycoside antibiotics have been widely used as important anti-infection drugs (Zaŕate et al., 2018).They usually have good intrinsic activity against gram-negative and some grampositive bacteria, killing them by binding to bacterial ribosomes and inhibiting normal protein synthesis (Kudo and Eguchi, 2016).Previous studies have shown that bacterial resistance to aminoglycoside antibiotics is usually driven by three main mechanisms: inactivation of the drug by aminoglycosidemodifying enzymes (AMEs), efflux, and targeted modification (e.g., by methylases) (Dozzo and Moser, 2010;Bastian et al., 2022).
Aminoglycoside O-phosphotransferases (APHs) are AMEs that utilize ATP or GTP to mediate the catalytic transfer of a phosphate group to an aminoglycoside molecule and make the antimicrobials inactivated.There are 35 functionally characterized APHs in the database CARD, and they are classified into 7 subfamilies [APH (2"), APH(3'), APH(3"), APH(4), APH(6'), APH(7") and APH(9)] (Alcock et al., 2023).Located over chromosomes and plasmids, they are sometimes associated with mobile genetic elements (MGEs), such as transposons, integrons and conjugative elements, which made them spread between bacteria of different species or genera by means of horizontal gene transfer (Toth et al., 2010).
Including 21 species, Citrobacter is a genus of the family Enterobacteriaceae.The bacteria of this genus have been clinically reported to cause central nervous system infections and sepsis in neonates and immunocompromised hosts (Doran, 1999).The species Citrobacter gillenii (type strain ATCC 51117 = CCUG 30796 = CIP 106783 = DSM 13694) of the genus was characterized by DNA hybridization and biochemical analysis in 1999 (Brenner et al., 1999).In the recent years, it has been increasingly isolated from aquaculture animals, where it has been found to carry multidrug resistance (MDR) genes which became a challenge for the treatment of farmed animals (Duman et al., 2017;Concha et al., 2021;Türe et al., 2022).
In this study, based on the whole genome sequencing, a novel aminoglycoside O-phosphotransferase gene, designated aph(3')-Ie, was identified in the chromosome of an animal isolate C. gillenii DW61, and its molecular and functional characteristics were further investigated.

Bacteria and plasmids
C. gillenii DW61 was isolated from an anal swab of an Oryctolagus cuniculus f. domesticus from a farm in Wenzhou, Zhejiang Province, China.The anal swab sample was streaked onto a standard Luria-Bertani agar plate, and single colonies were then isolated and purified by the same method.Species classification was first performed by 16S rRNA gene homology comparison (Wachino and Arakawa, 2012) and then confirmed by average nucleotide identity (ANI) calculations (Lindsey et al., 2023) and DNA-DNA hybridization (isDDH) analysis (Meier-Kolthoff et al., 2014).The strains and plasmids used in this study are listed in Table 1.

Drug susceptibility testing
The minimum inhibitory concentration (MIC) was tested by the plate dilution method using Mueller-Hinton (MH) agar (Thermo Fisher Scientific Inc. Beijing, China) in accordance with the most recent M100 performance standards for antimicrobial susceptibility testing by the Clinical and Laboratory Standards Institute (CLSI, 2024).After 16-20 h of constant incubation at 37°C , the results were interpreted following the CLSI M100 performance standards and the guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2022).Escherichia coli ATCC 25922 was used for a quality control.The experiments were conducted in triplicate.
Cloning of the aph(3')-Ie gene PCR was performed using primers to amplify the ORF of aph(3')-Ie with its upstream promoter region predicted by BPROM (www.softberry.com)(Table 2).The PCR product was subsequently inserted into the T-Vector pMD ™ 19 using T4 ligase (Takara Biomedical Technology Co., Ltd.).The recombinant plasmid in the ligation mixture was then transformed into competent E. coli DH5a cells by chemical transformation (Koppel et al., 2017).After screening colonies using an agar plate containing 100 mg/mL ampicillin, the sequence of the cloned fragment was verified by Sanger DNA sequencing (Shanghai Sunny Biotechnology Co., Ltd., Shanghai, China).

Primer a
Sequence (5' → 3') Vector Restriction endonuclease   C for 16 h.Bacteria were collected by centrifugation at 10,000 × g for 4 min, resuspended in 4 mL of nondenatured lysate, and immediately sonicated for 10 min.The lysis products were subsequently centrifuged at 10,000 × g for 30 min at 4°C.The supernatant was collected and reequilibrated with nickel-nitrilotriacetic acid (Ni-NTA) agarose resin (Beyotime Biotechnology, Shanghai, China).The mixture was gently agitated for 10 h at 4°C.Standard Ni-NTA affinity chromatography was used to isolate the His-tagged recombinant protein, and the His tag was removed using thrombin for 6 h at 37°C.The purified protein APH(3')-Ie was validated using SDS-PAGE, and the protein concentration was determined with the BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China).

Enzyme kinetics analysis
The kinetic parameters of APH(3')-Ie were measured in accordance with the previously reported methodology (Lu et al., 2021;Sha et al., 2023), with minor modifications.In brief, coupling between the production of ADP resulting from aminoglycoside phosphorylation and NADH oxidation was achieved through the use of pyruvate kinase (PK) and lactate dehydrogenase (LDH).The mixing volume for the entire reaction was 250 mL, which included 1 mM phosphoenolpyruvate (Beijing Solarbio Science & Technology Co., Ltd.), 600 mM NADH, 1 mM ATP, 100 mM HEPES (pH 7.0), 1 mM MgCl 2 , 2 mM KCl, a commercial mixture of PK and LD (Sigma

Identification of the novel resistance gene and classification and molecular characterization of the isolate DW61
To elucidate novel resistance mechanisms exhibited by bacterial isolates from local animals in response to antimicrobial agents, a total of 576 bacteria isolated from anal fecal swabs of domestic avians and livestock and environment in Wenzhou, China, were subjected to sequencing analysis (Feng et al., 2022;Gao et al., 2022;Zhao et al., 2023).Annotation of the genome data revealed the presence of resistance genes against various classes of antibiotics.Notably, among the predicted genes were some putative aminoglycoside antibiotic resistance genes, including but not limited to aph(3')-Ia, ant(9)-Ia, aadA5, aph(6)-Ic, aac(3)-IIIb, aac     S1.
(6')-Iaa, aph(6)-Id, and aac(2')-IIb homologues.These genes shared amino acid sequence identities below 80.0% with functionally characterized aminoglycoside resistance genes.Some of the genes were randomly selected and further cloned, and their resistance functions were determined.Ultimately, a novel aph(3')-Ia homologous gene (designated aph(3')-Ie in this work) that confers resistance to several aminoglycoside agents was identified from an isolate named DW61.

Distribution of the aph(3')-Ie genes and structural characterization of the aph(3')-Ie-related sequences
To analyze the distribution of the aph(3')-Ie gene and its close relatives, the amino acid sequence of APH(3')-Ie was used as a query to search for homologous protein-encoding nucleotide sequences using the tBLASTn program against the NCBI nucleotide collection (nt) database (Figure 4), and a total of 28 sequences showing ≥ 98.15% amino acid sequence identity with APH(3')-Ie were found in the database.The three strains with the highest identities (100.0%) were all C. freundii strains (including C. freundii RHBSTW-00714, CP056333.1;C. freundii RHBSTW-00006, CP056910.1;and C. freundii RHBSTW-00334, CP056597.1).Among the remaining 25 sequences, one was from the uncultured bacterium pJM6 from a metagenomic library of an environmental sample (FJ537710.1,identity 98.89%) and one was from Citrobacter werkmanii NBRC 105721 (LR699014.1,identity 98.15%), while the other 23 genes were also from C. freundii.Of the 28 genes, one was from a metagenomic library of an environmental sample, while the other 27 were from strains isolated from different sources, including animals (92.59%, 25/27), the environment (3.70%, 1/27) and humans (3.70%, 1/27) (Supplementary Table S2).
Further analysis of the sequences with lower identities revealed that of the 100 sequences with identities ≥ 70.94%, which included the 28 sequences mentioned above, 72 sequences had identities between 74.91% and 70.94%, and no sequence with an identity between > 74.91% and < 98.15% was present.Notably, of these 72 sequences with identities ranging from 70.94% to 74.91%, three sequences were from Klebsiella pneumoniae, including two encoded on plasmids (pBWH77 of K. pneumoniae 5214773, NG_047431.1,and pBWH77 of K. pneumoniae, X62115.1) and one in the chromosome (K.pneumoniae GDKA1, NG_047441.1),while the others were from other orders of bacteria, cloning vectors or synthetic constructs (Supplementary Table S2).
When analyzing the structure of the aph(3')-Ie gene-related sequences, a nucleotide sequence approximately 20 kb in length with the aph(3')-Ie gene at the center from the C. gillenii DW61 chromosome was used as a query to search for homologous sequences in the NCBI nucleotide database.A total of 28 sequences with identities ≥ 80.0% were retrieved, and they were all from the genus Citrobacter.Of these 28 sequences, 27 carried one aph(3')-Ie (-like) gene each and were from C. freundii, except one from C. werkmanii, MGYG-HGUT-02535 (LR699014.1,identity 98.41%).One sequence free of an aph(3')-Ie-like gene was from another species, Citrobacter ructae SNUWT2 (CP038469.1),and it shared a lower identity (81.62%) with that of C. gillenii DW61.No mobile genetic elements (MGEs) were detected within these 20 kb fragments.

Conclusion
In this study, a novel aminoglycoside phosphotransferase gene, designated aph(3')-Ie, was identified in the chromosome of the MDR Citrobacter gillenii isolate DW61, which showed high MICs (≥ 16 mg/mL) for 76.7% of the 43 antibiotics tested.This novel resistance gene conferred resistance to some aminoglycosides, such as ribostamycin and kanamycin, and was present in C. gillenii from different sources, including some of clinical significance.The discovery of new antimicrobial resistance mechanisms is helpful for effective clinical treatment of bacterial infectious diseases.

a
Primers ending with "clo" were used to clone the ORF of the aph(3')-Ie gene and its promoter region; primers ending with "exp" were used to clone the ORF of the aph(3')-Ie gene.Restriction enzyme sites are underlined.
DH5a was used as the host bacterium for cloning of the aph(3')-Ie gene with its upstream promoter region CGMCC* ATCC 25922 Pseudomonas aeruginosa ATCC 25922 was used as a quality control strain for antimicrobial susceptibility testing CGMCCDH5a carrying the recombinant plasmid pMD19-Taph(3')-Ie This study DH5a(pMD19-T) E. coli DH5a carrying the pMD19-T vector was used as a control strain in the drug susceptibility testing CGMCC* BL21(pCold I-aph(3')-Ie) E.coli BL21 carrying the recombinant plasmid pCold I-aph(3pMD ™ 19 (Simple) was used as a vector for cloning of the aph(3')-Ie gene with its upstream promoter region, AMP r CGMCC* pCold I pCold I was used as a vector for expression of the ORF of the aph(3')-Ie gene, AMP r CGMCC* *CGMCC, China General Microbiological Culture Collection Center.

FIGURE 1
FIGURE 1The genome map of C. gillenii DW61.The circles 1-4 from inside to outside represent the GC skew, GC content, and genes encoded in the forward and reverse strands of the chromosome of C. gillenii DW61, respectively.The red line on the bottom indicates the location of the novel resistance gene aph(3')-Ie.

FIGURE 2 A
FIGURE 2 A phylogenetic tree showing the relationships of APH(3')-Ie with other functionally characterized APH(3')s.APH(3')-Ie is highlighted in red.The accession numbers of these proteins are listed in Supplementary TableS1.

FIGURE 3
FIGURE 3 Multiple-sequence alignment of APH(3')-Ie with its close relatives.The numbers on the right represent the corresponding amino acid sequence lengths.The red frames represent the residues of the conserved APH protein domain family.The exclamation marks indicate the residues of the ATP-binding sites, and the asterisks indicate the 12 residues of the antibiotic-binding site.The conserved residues are shown in dark blue, while the relatively conserved residues are shown in light blue.Gaps are represented using hyphens.

FIGURE 4
FIGURE 4 Genetic background of the aph(3')-Ie gene and the aph(3')-Ie homologous genes.Regions with an amino acid identity of ≥ 80% are in blue.hp, hypothetical protein.The aph(3')-Ie and aph(3')-Ie-like genes (similarity > 98.15%) are shown in red.The aph(3')-Ia gene is shown in green, and the hypothetical protein-encoding genes are shown in blue.

TABLE 1
Strains and plasmids used in this work.

TABLE 3
MIC results of the recombinants and control strains (µg/mL).The velocities at steady state were determined by analyzing the linear phase of the reaction progress curve and then plotted as a function of the substrate concentration.The Michaelis-Menten equation was used to fit the data through nonlinear regression, with velocity as a function of substrate.GraphPad Prism 8.0.2 (GraphPad Software, CA, United States) was used for curve fitting and calculation of the dynamic parameters K m and k cat .

TABLE 3 Continued
"/" the test was not performed.

TABLE 4
General features of the DW61 genome.